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Regulation of acetate kinase isozymes and its importance for
mixed acid fermentation in Lactococcus lactis
Pranav Puri1, Anisha Goel2, Agnieszka Bochynska1 and Bert Poolman1
1Department of Biochemistry, Groningen Biomolecular Sciences and Biotechnology
Institute, Netherlands Proteomics Centre & Zernike Institute for Advanced Materials
University of Groningen, Nijenborgh 4, 9747 AG Groningen, The Netherlands
2Cell Systems Engineering section, Department of Biotechnology, Delft University of
Technology, Julianalaan 67, 2628 BC Delft, The Netherlands
Correspondence to Bert Poolman
Email: [email protected]
Tel.: +31 50 3634209
Key words: Lactococcus lactis, mixed acid fermentation, acetate kinase, allosteric
regulation, enzymology
Running title: Actetate kinases from L. lactis
JB Accepts, published online ahead of print on 24 January 2014J. Bacteriol. doi:10.1128/JB.01277-13Copyright © 2014, American Society for Microbiology. All Rights Reserved.
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Abstract
Acetate kinase (ACK) converts acetyl phosphate to acetate along with the generation of
ATP in the pathway for mixed acid fermentation in Lactococcus lactis. The reverse
reaction yields acetyl-phosphate for assimilation purposes. Remarkably, L. lactis has two
ACK isozymes and the corresponding genes are present in an operon. We purified both
enzymes (AckA1 and AckA2) from L. lactis MG1363 and determined their oligomeric
state, specific activity and allosteric regulation. Both proteins form homodimeric
complexes as shown by size-exclusion chromatography and static light-scattering
measurements. The turnover number of AckA1 is about an order of magnitude higher
than that of AckA2 for the reaction in either direction. The Km values for acetyl
phosphate, ATP and ADP are similar for both enzymes. However, AckA2 has a higher
affinity for acetate as compared to AckA1, suggesting an important role under acetate-
limiting conditions despite the lower activity. Fructose-1,6-bisphosphate, glyceraldehyde-
3-phosphate and phosphoenolpyruvate inhibit the activity of AckA1 and AckA2 to a
different extent. The allosteric regulation of AckA1 and AckA2 and the pool sizes of the
glycolytic intermediates are consistent with a switch from homolactic to mixed acid
fermentation upon lowering of the growth rate.
Introduction
Lactococcus lactis is a Gram-positive, anaerobic, non-spore forming bacterium
that produces lactic acid from glucose or lactose when grown in batch culture. This
homolactic fermentation is used as a trait in the biotech industry, as acidification of the
environment is an important parameter in the preservation and shelf-life of food products
(1, 2). L. lactis lacks a heme biosynthesis pathway (3). However, if L. lactis is grown in
the presence of heme in the culture medium, it can synthesize a minimal electron transfer
chain and form a proton motive force by a simple form of respiration (Brooijmans et al.,
2009). This increases the biomass production and robustness of bacteria (4-6). However,
under most conditions the (energy) metabolism of L. lactis is strictly fermentative.
Depending on the availability of carbohydrate source, the fermentation can be homolactic
or mixed-acid. Mixed acid fermentation yields an additional ATP and acetic acid, formate
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and ethanol as end-products (7, 8) (Fig. 1). The exact mechanism responsible for the shift
from homolactic to mixed acid fermentation and vice versa is not well understood. In
fact, in one of the hypotheses the metabolic shift is proposed to be controlled by fructose
1,6-bisphosphate (FBP) (7, 9). The concentration of FBP increases with increasing
glucose flux and the metabolite has been shown to be an allosteric activator of pyruvate
kinase (PK) (10, 11) and lactate dehydrogenase (LDH) (7). During starvation the
concentration of FBP decreases, which reduces PK and LDH activity and thereby the
glycolytic flux. In L. lactis lysates, FBP has been shown to inhibit acetate kinase (ACK)
and phosphotransacetylase (PTA) (12). Other metabolites whose concentrations
significantly change during growth of L. lactis in carbon-limited medium are
dihydroxyacetone phosphate (DHAP) and glyceraldehyde-3-phosphate (GAP). DHAP
and G3P are allosteric inhibitors of pyruvate formate lyase (PFL), the enzyme that
catalyzes the reversible conversion of pyruvate and coenzyme-A to formate and acetyl-
CoA. In principle, a decrease in concentration of these triose phosphates could result in a
metabolic shift to mixed-acid fermentation products (13, 14). Besides these glycolytic
intermediates an important role is attributed to the NADH/NAD+ ratio, which has been
shown to control the flux through both glyceraldehyde-3-phosphate dehydrogenase
(GAPDH) and LDH. While GAPDH activity is low at high NADH/NAD+ ratio, the
activity of LDH is positively modulated by high NADH/NAD+ (15). Other mechanisms
of regulation of the fermentation shift include changes in expression level of specific
genes (pfl) (16) and the las operon (15), the latter encodes phosphofructo kinase (PFK),
pyruvate kinase (PK) and lactate dehydrogenase (LDH). In a ‘multi-omics’ analysis on L.
lactis grown at different growth rates in a chemostat, relatively small changes in the
proteome during the shift from mixed acid to homolactic fermentation [Goel et al,
unpublished] were observed, which suggests that expression regulation is insufficient to
explain the metabolic shift.
Acetate kinase (ACK) converts acetyl phosphate to acetate along with the
phosphorylation of ADP and is one of the most prominent ATP-generating reactions in
anaerobic microorganisms. Acetate kinase [EC 2.7.2.1] was first discovered 70 years ago
in extracts from Lactobacillus delbrueckii but the activity has been described for
facultative and obligate anaerobes (17, 18), incl. Escherichia coli, Salmonella
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typhimurium (18), and various thermophilic bacteria (19, 20). The reaction catalyzed by
acetate kinase is reversible and requires monovalent (K+) and divalent (Mn2+or Mg2+)
cations. The equilibrium lies far towards ATP formation with a equilibrium constant in
the range from 200 to 1500 (21). Despite its importance microbial metabolism the
regulation of acetate kinase activity is poorly documented.
It is not common for organisms to have more than one gene encoding acetate kinase, and
the enzyme has been described as homodimer in E. coli (18) and Methanosarcina
thermophila (19) and homotetramer in Bacillus stearothermophilus (22). In L. lactis
subsp. cremoris two isozymes of acetate kinase are present. The loci of both genes lie
very near to each other and are thought to be under control of the same promoter. Both
genes are 1188 base pairs long and have very high sequence similarity at the nucleotide
and amino acid level (67% identity). The current study is aimed at a better understanding
of the enzymology of the two acetate kinases of L. lactis and the role of the two enzymes
in the regulation of the metabolic shift, i.e. from homolactic to mixed acid fermentation.
Materials and methods
Ligation-independent cloning (LIC)
Expression vectors used for cloning and expression of acka1 and acka2 were pBADnLIC
and pBADcLIC, and E. coli MC1061 was used as expression host (23). Plasmid DNA
was purified from E. coli by the Miniprep kit (Qiagen). The sequences of acka1 and
acka2 were taken from the NCBI database and used to design the primers for
amplification of the genes by the polymerase chain reaction (PCR). The oligonucleotide
primers 5’ATTGGCTAGTCTGTCAGTA3’ and
5’CACAAGAAGAATTCCGGCAATCC3’ were used to amplify both genes from L.
lactis subspecies cremoris MG1363 genomic DNA in a single template. Primers (0.5
μM), dNTPs (200 μM), 1 U of PhusionTM DNA polymerase (New England Biolabs) and
20 ng of DNA were used in a reaction volume of 50 μl. PCR reaction cycles included
denaturation of the DNA at 98 C for 5 min, followed by 35 cycles of 98 C for 20 s, 55 C
for 20 s, and 72 C for 90 s, with a final cycle of 72 C for 5 min. PCR amplification
products were purified from agarose gels (1%), electrophoresis in TAE buffer, and
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extracted from the gel by the PCR cleanup and gel extraction kit (Macherey Nagel). The
template so obtained was digested by BglII (NEB) to separate acka1 and acka2.
Digestion was performed in 20 μl reaction volumes containing amplified long DNA
fragment (15 μl), NEB buffer 3 (2 μl) and BglII (1 U, New England Biolabs), and the
mixture was incubated at 37 C for 60 min. Next, primers were designed for Ligation-
Independent Cloning of acka1 and acka2 in pBADnLIC and pBADcLIC (Table 1).
Amplification reactions were performed in 50μl volume, containing restriction digestion
products as template DNA, each primer (0.5 μM), dNTPs (200 μM) and 1 U of
PhusionTM DNA polymerase (New England Biolabs). PCR and purification of the DNA
was done as described above. T4 polymerase-treatment of vector and insert was
performed in 15 μl reaction volume, containing 250 μM dCTP/dGTP, 200 ng of vector
and T4 polymerase (1U, Roche), and the mixture was incubated at room-temperature for
30 min as described previously (24). The ligation of vector and insert was performed in
12-μl reaction volume, containing vector to insert at a ratio of 1 to 3, and the mixture was
incubated at room temperature for 5 min and then transformed to competent E. coli
MC1061, according to the standard protocol (25).
Optimization of protein expression
5 ml of E. coli culture induced with 0.01% (w/v) arabinose for 2h was resuspended in ice-
cold 50 mM KPi (pH 7.0) with 10% glycerol, 1 mM magnesium sulfate, 0.1 mg/ml
DNAse, 1 mM PMSF and 300 mg of glass beads (0.1 μm diameter). Cells were ruptured
for 5 min at 50 Hz in a tissue-lyser (Qiagen). To the cell lysate was added 5 mM EDTA
to minimize protein degradation and aggregation. Samples were centrifuged for 15 min at
16,000 x g in a tabletop centrifuge, 4 C. Proteins were separated by SDS-PAGE and
analyzed by Western blotting, using an antibody against the His-tag epitope and
visualized by chemi-luminescence of alkaline phosphatase in the Fujifilm LAS-3000
Imager.
Protein purification and molecular mass determination
Cells expressing either AckA1 or AckA2 were grown at 37°C in 1L of LB medium (1.0%
Tryptone, 0.5% Yeast Extract, 1.0% Sodium Chloride (NaCl)) and 100 μg/ml Ampicillin.
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Expression in E. coli was induced at an OD600 of 0.4 by the addition of 0.01% (w/v) L-
arabinose. E. coli cells harboring pBADcLIC acka1 were grown at 37°C, while the ones
harboring pBADcLIC acka2 were grown at 37°C and 25°C. Upon induction for 2h, the
cells were harvested by centrifugation at 6500 x g for 15 min and resuspended in 40 ml
50 mM KPi pH 7 with 1 mM PMSF. Bacteria were disrupted in the Constant Systems TS
(Biosystems) upon addition of 1 mM magnesium sulfate and 0.1 mg/ml RNase/DNase to
the resuspended cells. To the crude lysate were added 5 mM EDTA. The lysate was
centrifuged at 15,000 x g for 15 min (4 C), and the supernatant was used for purification
of the enzymes. Affinity chromatography was carried out at 4 C. The cell extract (~ 25
mg of total protein) was applied to the column containing 500 l of bed volume. Ni-
Sepharose equilibrated with 50mM KPi, pH 7 and 150 mM NaCl. Columns were washed
with 20 ml of equilibration buffer with 10 mM of imidazole. Enzymes were eluted by an
imidazole gradient from 50-500 mM in 50 mM KPi, pH 7 plus 150 mM NaCl. Samples
from each step were collected and analyzed by SDS-PAGE. Fractions with the highest
ACK concentration were applied onto a Superdex 200 10/300GL gel filtration column
(GE Healthcare) and eluted at a flow rate of 0.4 mL/min with 50 mM KPi pH 7 plus 150
mM NaCl, using an Agilent 1200 series isocratic pump at 4oC. The oligomeric state of
AckA1 and AckA2 was determined by size-exclusion chromatography coupled to multi-
angle laser light scattering (SEC-MALLS) as described before (26, 27). 200 l (150 g)
of purified protein was used in the experiment. The method described by Wen et al was
used to estimate the molecular mass of the native complexes (28). The molecular mass
(MW) of the protein was calculated using the classical Rayleigh relationship (29):
LS = (I /I )solution – (I /I )buffer = K(dn/dc)2 MWC
LS is the excess of light scattered at a given angle by the solution containing the
protein and compared to the buffer without the protein. I /I is the ratio of the intensities
of the light scattered at angle and the incident light. MW is the molecular mass of the
scattering protein. C is the concentration of protein (in mg/ml), which is directly
measured using a concentration sensitive detector. dn/dc is the change in refractive index
of the solution with respect to the protein concentration. K is a constant, which depends
on the refractive index of the solution without macromolecule (n), the wavelength of the
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light used ( ), the angle between the incident and scattered light ( ) and the distance
between the scattering molecule and the detector (r) and is calculated as:
K = 2 2n2 / 4NA/ ((1+cos2 )/r2), where NA is Avogadro’s number.
Protein determination
The concentration of AckA1 and AckA2 was determined by the NanoDrop device, using
extinction coefficients calculated on the basis of the amino acid composition of the
proteins. The extinction coefficient (ε) of AckA1 and AckA2 are 28,880 M-1 cm-1 and
27,390 M-1 cm-1, respectively.
Enzyme assays
Enzyme assays were carried out at 30 C. For the reaction in the direction of ATP and
acetate formation, the assay mixture consisted of potassium-HEPES pH 7.5 (0.1 M),
potassium phosphate tribasic (1 mM), sodium chloride (50 mM), magnesium sulfate
heptahydrate (5 mM), hexokinase (2.5 U), glucose-6P-dehydrogenase (2.3 U), glucose
anhydrate (2 mM), ADP (3 mM), NADP+ (0.4 mM) and acetyl-phosphate (5 mM), unless
specified differently (addition of glycolytic intermediates). Reactions were performed in
a total volume of 300 μl (SynergyMx Plate Reader, BioTek). The reaction was initiated
with 30 μl of 0.05 M acetyl phosphate or 18 μl of 50 mM ADP. For the enzymatic
reaction in the direction of acetyl phosphate formation, the assay mixture consisted of
potassium-HEPES pH 7.5 (0.1 M), potassium phosphate tribasic (1 mM), sodium
chloride (50 mM), 4.2 mM magnesium chloride, 1.7 mM phosphoenolpyruvate, 0.24 mM
NADH, 10 units of PK/LDH cocktail (Sigma) and varying amounts of ATP and Acetate.
The coupled enzyme assay was performed as described in Goel et al (30). The Substrate
concentrations and rates were fitted to the Michaelis–Menten equation, or a modified
form: V=Vmax*X/[Km + X*(1+X/Ki)], using Graph pad Prism 5, to estimate Km, Ki and
Vmax values. The effect of crowding was tested by adding PEG 6000, 4000, 1500 or 200
at a concentration of 0-10 % (w/v) to the assay mixture.
Results
Overexpression, purification and oligomeric state
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The two isozymes of acetate kinase in L. lactis, acka1 and acka2, were separately cloned
and overexpressed in E. coli. The acka1 and acka2 gene from L. lactis subsp. cremoris
MG1363 was inserted in the SwaI restriction digestion site of pBadNlic and pBadClic.
Using these plasmids, the recombinant expression of acka1 and acka2 results in
translation products with a 10His-tag at either the N- or C-terminus of the protein; the
synthesis is under the control of the L-arabinose promoter. The expression of both
enzymes was tested at two different temperatures (25 C and 37 C) and three different
concentrations of L-arabinose (0.01, 0.001 and 0.0001% w/v). Both proteins expressed
well when induced with 0.01% (w/v) L-arabinose at 37 C. In general, the C-terminal
tagged version expressed better than the N-terminal one (data not shown), and hence the
C-terminal his-tagged ACKs were used for most experiments.
Purification of AckA1 and AckA2 was carried by Ni-Sepharose affinity chromatography,
and the proteins were eluted in three fractions (Fig. 2B and D; lanes 1, 2 and 3). To
further purify the proteins, the elution fraction 2 was subjected to size-exclusion
chromatography (SEC) and the resultant protein fractions are indicated in Fig 2B and D;
lanes 4. The 2-step purified protein was used for further characterization as described in
the sections below. The yield from 1 l of cell culture at O.D600 = 2.5 was 3-4 mg of
protein for both AckA1 and AckA2. To determine the oligomeric state of AckA1 and
AckA2, the proteins were subjected to SEC coupled to Multi-angle laser light scattering
(SEC-MALLS). The SEC-MALLS profiles are shown in Fig. 2A and C. The molecular
mass of each peak in the static light scattering was calculated as described in materials
and methods. The molecular mass profile of both AckA1 and AckA2 showed a relatively
homogenous distribution around 13 ml, which corresponds to a molecular mass of 93
kDa. Since the calculated molecular mass of both proteins is 43 kDa, the native enzyme
complexes are dimeric
Enzymatic activity
The activity of acetate kinase for the catalytic generation of ATP has been determined in
crude cell lysate in which contributions from AckA1 and AckA2 and possibly other
enzymes could not be discriminated (12). We determined the activities of the purified
enzymes in media mimicking the interior of the cell in terms of pH and ion composition
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but low in (macro)molecular crowding. For the ATP generation reaction, AckA1 is an
order of magnitude more active than AckA2 (Fig. 3). We also mixed the two enzymes in
different ratios viz 1:1, 1:4 and 4:1 (AckA1:AckA2) to determine whether in solution they
would possibly form heterodimeric complexes or influence each other via higher order
complex formation. In all the cases where a combination of the two enzymes was used,
the combined activity was equal to the sum of individual activities (Fig. 3).
Acetate kinase catalyzes the transfer of phosphate from acetyl-phosphate to ADP to form
ATP and acetate. We thus determined the Km and Vmax values as function of the
concentration of acetyl-phosphate and ADP. The kinetic parameters for acetyl phosphate
were determined by initiating the reaction with ADP and vice versa. At an ADP
concentration of 3 mM the Km values of AckA1 and AckA2 for acetyl phosphate were
nearly identical with values of 0.54 mM and 0.55 mM, respectively (Fig. 4, panels A to
D); the data are summarized in Table 2(A). The maximum velocity of AckA1 was about
an order of magnitude higher than that of AckA2. The turnover numbers (kcat) of AckA1
and AckA2 are 1100 and 100 s-1, respectively (Table 2).
By varying the ADP concentration and keeping acetyl-phosphate at 5 mM, we obtained
activities that leveled off around 2 mM; above this concentration both enzymes were
inhibited, which is indicative of substrate inhibition (Fig. 4, panels C and D). Because of
the non-Michaelis-Menten kinetics, the data were fitted to an equation that takes into
account substrate inhibition:
V=Vmax*X/[Km + X*(1+X/Ki)] Eq.1
in which Km, Ki and Vmax correspond to the affinity constant for X (=ADP), the inhibition
constant for X and the maximal rate of the reaction, respectively. The Km values of
AckA1 and AckA2 for ADP are 0.47 mM and 0.74 mM, respectively. The Ki (ADP)
values of Ack1 and Ack2 are 2.4 and 3.9 mM, respectively (Table 2A). The inhibition of
the ACKs by ADP is relevant at low growth rate and under conditions of energy
starvation when the ADP concentrations are in the mM range; in fast growing cells the
ADP concentrations are well below 1 mM (31). To assess whether or not the inhibition
by ADP was not due to chelation of magnesium ions, the activity of AckA1 was
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determined by varying the ADP concentration in the presence of 5 and 30 mM MgCl2.
Both under conditions of magnesium or ADP excess, AckA1 was inhibited by ADP at
concentrations >2 mM (Fig. 5).
L. lactis can assimilate acetate but cannot grow on acetate as sole energy/carbon source.
We thus investigated the kinetic properties of the two enzymes in the direction of acetyl
phosphate formation. AckA1 also showed an order of magnitude higher activity in the
reaction form acetate plus ATP to acetyl phosphate plus ADP (Fig. 4 panels E to H). The
Km values for ATP were about 0.07 mM for both enzymes. However, the Km of AckA1
for acetate (20.5 mM) was 4 to 5-fold higher than that of AckA2 (4.9 mM). The kinetic
parameters for the reaction in the direction of acetyl phosphate formation are summarized
in Table 2B.
Effect of crowding
The activities of enzymes in vitro can be very different from the ones in vivo due to the
high crowding conditions (excluded volume effects) in the cytoplasm (32). We tested the
activity of AckA1 in the presence of varying concentrations (from 0 to 10 % (w/v)) of
different size polyethylene glycols (PEG 200, 1500 and 4000 and 6000) as crowding
agents. PEGs can stabilize or destabilize a protein, depending on the size and
concentration of the molecules (see Discussion). The specific activity of AckA1
decreased by 20-30 % and plateaus at a concentration of about 4 % (w/v), irrespective of
whether a low or relatively high molecular weight PEG was used (Fig. 6). The effect of
varying concentrations of PEG 6000 was also tested on AckA2, which yielded a similar
dependence as for AckA1 (data not shown). We thus conclude that AckA1 and AckA2
are not particularly sensitive to crowding conditions.
Regulation of AckA1 and AckA2
Fructose 1,6-bisphosphate (FBP) has been shown to inhibit acetate kinase activity in L.
lactis lysate (7). We now show that FBP inhibits both AckA1 and AckA2. We tested FBP
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up to a concentration of 100 mM under conditions that the enzymes are partially inhibited
by ADP (3 mM) and saturated with acetyl-phosphate (5 mM). For AckA1 the IC50
(inhibitor concentration at 50 % activity) for FBP was 17 mM (Fig. 7A and B), while for
AckA2 the IC50 was 43 mM. In order to elucidate the mechanism of the inhibition, we
determined the activity of the enzyme at 0, 17 and 50 mM FBP. We observe a decrease in
Vmax with increasing FBP was while the Km for acetyl phosphate was not significantly
affected. This is consistent with a non-competitive mode of enzyme inhibition (Fig. 8).
Glyceraldehyde-3-phosphate (G3P) and dihydroxyacetone phosphate (DHAP) are formed
upon cleavage of FBP. While DHAP had no effect (data not shown) on the activity of
both acetate kinases, G3P inhibited the activity of AckA1 with an IC50 of 4 mM (Fig. 7C
and D). The activity of AckA2 was much less affected by G3P, and only at non-
physiological concentrations a maximum inhibition of 50 % was observed.
Phosphoenolpyruvate, a downstream intermediate of glycolysis, completely inhibits the
activity of both enzymes above 30 mM. The IC50 values for PEP for AckA1 and AckA2
were 15 and 18 mM (Fig. 7E and F). Next, we investigated the inhibition of AckA1 by
FBP, PEP and G3P at ADP and acetyl phosphate concentrations near the Km for both
substrates (about 0.5 mM). The IC50 values for FBP, PEP and G3P remained unchanged,
confirming that substrates do not influence the binding of inhibitors to the regulatory site
on the enzyme.
Discussion
ACKs catalyze the synthesis of ATP from acetyl phosphate and ADP, leading to the
formation of acetate. The enzyme can be also used in the reverse reaction but
thermodynamically the equilibrium is towards ATP formation (21, 33). L. lactis subsp.
cremoris MG1363 is one among many bacteria that has two or more genes for ACK. All
the active sites residues described for ACK from Methanosarcina thermophila (34) and
Salmonella typhimurium (35), whose crystal structures are known, are conserved in the
two enzymes from L. lactis MG1363. The ACKs characterized so far are oligomeric
enzymes and the majority forms dimer complexes (including the enzymes from M.
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thermophile (34), Salmonella typhimurium (18, 35) and E. coli (18)). The enzyme from
Bacillus stearothermophilus is described to be homotetrameric (22). The kinetic
parameters of the acetate kinase from S. typhimurium (18, 35), E. coli (18), Clostridium
acetobutylicum {Winzer:1997fn} and Thermotoga maritima (20) have been determined
for both the forward and reverse reaction. The Km for acetate varies from 0.07 mM for
the enzymes in E. coli and S. typhimurium to 73 mM for the enzyme from C.
acetobutylicum. The Km values for ATP vary from 0.37 mM (C. actetobutylicum) to 7
mM (S. typhimurium). The values for Km(ADP) and Km(Acetyl-P) are below 1 mM for all
described acetate kinases.
We determined the kinetic parameters of AckA1 and AckA2 for both reaction directions.
The turnover number (kcat) of AckA1 is an order of magnitude higher than that of AckA2
in both reaction directions (Table 2), and both enzymes are inhibited by millimolar
concentrations of ADP. The inhibition by ADP is relevant under conditions of slow
growth or energy starvation when ADP concentrations are in the millimolar range (about
5 mM) (36). The Km values of AckA1 and AckA2 for acetyl phosphate are nearly
identical. However, the Km of AckA2 for acetate was 4 to 5-fold lower than that of
AckA1. This implies that the relative contribution to the formation of acetyl phosphate of
AckA2 is highest at low acetate concentrations. Thus, while AckA1 is the preferred
enzyme for ATP formation; the role of AckA2 is primarily in acetate assimilation and
synthesis of acetyl-phosphate. In a recent study, Weinert et al, demonstrated increased
acetylation of metabolic enzymes in E. coli when cells enter the stationary phase. The
increased acetylation stems from the generation of acetyl phosphate (37, 38). It is
possible that the different catalytic efficiencies (kcat/Km) of AckA1 and AckA2 in the
forward and backward reactions tune the pools of acetyl phosphate and thus protein
acetylation and contribute to the metabolic shift in L. lactis.
The cell environment is crowded with molecules like proteins and nucleic acids (39, 40).
The activities of enzymes that are determined in vitro can significantly differ from the
ones inside a cell, owing to the high crowding and/or relatively low water activity of the
cytoplasm. We thus determined the effect polyethylene glycols (PEGs) as crowding
agents on the activity of the ACKs. PEGs are artificial crowding agents that can alter the
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stability/conformation of proteins depending on their molecular mass and concentration.
PEG molecules can have destabilizing and stabilizing effects on proteins. Destabilizing
effects of PEG molecules originate from the Lifshitz-electrodynamic interaction of the
co-solvent molecules with proteins, and these are attractive regardless of the chain length
of the PEG molecules (41, 42). The stabilization of protein structure by PEG molecules is
attributed to the steric excluded volume effects, which are caused by entropic forces of
the PEG molecules. The larger the PEG molecule the more the attractive forces are
overcome by the repulsive forces, i.e. the protein destabilization by binding of PEG
molecules is overcome by the excluded volume effects. Both AckA1 and AckA2 showed
a decrease in activity by 20-30% at PEG concentrations of 4-10 % (w/v). Since the
effects of low and high molecular weight PEGs are similar, we have no indications for
stabilizing/activating effects by high concentrations of (macro)molecules.
FBP has been implicated in the regulation of L. lactis metabolism by acting as allosteric
activator of lactate dehydrogenase (13) and inhibitor of acetate kinase (12). We now
confirm the inhibition of acetate kinase and find that both AckA1 and AckA2 are
inhibited by FBP. The intracellular FBP concentration in L. lactis cells grown on various
sugars in batch culture can vary from 25 to 118 mM (8). Neves et al, estimated
intracellular FBP at 50 mM during homolactic fermentation (43), a concentration at
which the residual activity of AckA1 and AckA2 would be less than 20 % and lactate
dehydrogenase would be maximally activated. This would indeed favor homolactic over
mixed acid fermentation. Intriguingly, we find that both acetate kinases are strongly
inhibited by PEP and the inhibition of AckA1 by G3P is more pronounced than that of
AckA1; DHAP had no effect on either of the enzymes. In fast, glucose- or lactose-
metabolizing cells the pools of G3P and DHAP are around 0.6 mM. In galactose-
metabolizing cells the pools of G3P and DHAP are even lower (8). We thus conclude that
the inhibition of AckA1 by G3P is most likely not physiologically relevant. On the other
hand, PEP caused inhibition of both ACKs with an IC50 of 15 and 18 mM for AckA1 and
AckA2, respectively. In L. lactis cells grown on glucose or galactose, the concentration
of PEP was 25mM and 0.6 mM, respectively. Thus, at high growth rates in the presence
of glucose, PEP may contribute significantly to the inhibition of acetate kinase, which
would favor homolactic fermentation.
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In conclusion: AckA1 is an order of magnitude more active than AckA2 in both
reaction directions, but Ack2 has the highest affinity for acetate which gives the enzyme
a more prominent role in the formation of acetyl phosphate at low substrate
concentrations. We show that major glycolytic intermediates allosterically control the
activity of AckA1 and AckA2. The reciprocal allosteric regulation of acetate kinases is a
mechanism that can allow L. lactis to switch almost instantaneously between homolactic
and mixed fermentation, which occurs as a function of the availability of fast or slow
metabolizable sugars.
Acknowledgements
This work is supported by the Dutch Technology Foundation STW (grant 08080), which
is part of the Netherlands Organization for Scientific Research (NWO). We acknowledge
the help of Dirk-Jan Slotboom with the SEC-MALLS measurements. BP is supported by
NWO-TOP GO subsidy (grant number 700.10.53). BP is additionally supported by the
Netherlands Proteomics Centre.
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Figure Legends
Figure 1. Schematic representation of glucose catabolism in L. lactis MG1363.
Figure 2. Static light-scattering profiles of AckA1 (A) and AckA2 (C) and SDS-PAA
(10%) polyacrylamide gel electrophoresis analysis of AckA1 (B) and AckA2 (D). Lanes
1, 2 and 3 in panel (B) and (D) represent the elution fractions after Ni-Sepharose
purification, while lane 4 represents the peak fraction after SEC. The grey line in (A) and
(C) indicates the molecular mass.
Figure 3. Acetate kinase activity for the reaction involving ATP formation at different
ratios of AckA1 and AckA2 as indicated in the figure. The experimental (Exp) and
calculated values (Cal), assuming independent functioning of the enzymes, are plotted.
Figure 4. Specific activity of AckA1 and AckA2 as a function of acetyl-phosphate
(panels A and B), ADP (panels C and D), acetate (panels E and F) and ATP (panels G
and H) concentration.
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Figure 5. Effect of magnesium ion concentration on substrate inhibition of AckA1 by
ADP. ( ) 5 mM; ( ) 30 mM magnesium chloride
Figure 6. Specific activity of AckA1 in the presence of varying concentrations of ( )
PEG 6000; ( ) PEG 4000; ( ) PEG 1500 and (×) PEG 200.
Figure 7. Effect of fructose-1,6-bisphosphate (A, B), glyceraldehyde-3-phosphate (C, D)
and phosphoenolpyruvate (E, F) on the specific activity of AckA1 ( ) and AckA2 ( )
Figure 8. Specific activity of AckA1 with Acetyl phosphate as substrate, in the presence
of ( ) 0 mM, ( ) 17 mM and ( ) 50 mM FBP.
Table 1. Primer sequences used for cloning of acka1 and acka2 in pBADcLIC and
pBADnLIC
Table 2. The values for the affinity constants and activities of AckA1 and AckA2 in the
direction of (A) ATP plus acetate and (B) acetyl phosphate plus ADP formation. The
parameters were obtained after fitting of the data to the Michaelis Menten equation w/wo
component for substrate inhibition.
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Primer Sequence (5’ – 3’)
AckA1 Forward for pBADnLIC
ATGGTGAGAATTTATATTTTCAAGGTATGACCAAAACATTAGCAGTAAAC
AckA1 Reverse for pBADnLIC
TGGGAGGGTGGGATTTTCATTATTTTTTAAGTGCCTCAACGTCGCGAGC
AckA1 Forward for pBADcLIC
ATGGGTGGTGGATTTGCTATGACCAAAACATTAGCAGTAAAC
AckA1 Reverse for pBADcLIC
TTGGAAGTATAAATTTTCTTTTTTAAGTGCCTCAACGTCGCGAGC
AckA2 Forward for pBADnLIC
ATGGTGAGAATTTATATTTTCAAGGTATGGAAAAAACGCTCGCTGTC
AckA2 Reverse for pBADnLIC
TGGGAGGGTGGGATTTTCATTATTTAGCCGCTTCGACATCACG
AckA2 Forward for pBADcLIC
ATGGGTGGTGGATTTGCTATGGAAAAAACGCTCGCTGTC
AckA2 Reverse for pBADcLIC
TTGGAAGTATAAATTTTCTTTAGCCGCTTCGACATCACG
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(A) Acetyl phosphate + ADP Acetate + ATP
Enzyme Km(Acetyl phosphate) (mM)
Km(ADP) (mM)
Ki(ADP) (mM)
Vmax ( mol.min-1.mg-1)
kcat (s-1)
AckA1 0.54±0.1 0.47±0.1 2.4±0.7 1526±74 1105±65
AckA2 0.55±0.1 0.74±0.2 3.9±1 129±5 93±6
(B) Acetate + ATP Acetyl phosphate + ADP
Enzyme Km(Acetate) (mM)
Km(ATP) (mM)
Vmax ( mol.min-1.mg-1)
Kcat (s-1)
AckA1 20.54±2.0 0.07±0.01 1051±34 761±34
AckA2 4.9±0.6 0.07±0.01 111±2 80±5
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